NO-dependent CFTR activation (normal and
Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene
encoding the CF transmembrane conductance regulatory (CFTR)
protein . The most common mutation associated with CF results in deletion
of a single amino acid, phenylalanine, at position 508 in
CFTR protein (mutant deltaF508 CFTR protein) , .
Exhaled nitric oxide (NO), elevated in most inflammatory
lung diseases, is decreased in CF. According to some studies, impaired
NO formation in lower airways correlates with more
progressive lung disease .
NO is produced by a family of NO synthases (NOS) by
transformation of L-arginine to L-citrulline and
NO. In human, there are three isoforms of NOS: neuronal NOS
(nNOS), endothelial NOS (eNOS)
and inducible NOS (iNOS). nNOS
and eNOS are constitutively expressed and produce small
amounts of NO, whereas iNOS
expression is mainly induced by inflammatory stimuli. Induced iNOS synthesizes relatively
large quantities of NO. All three isoforms are expressed in
human airways .
In the case of CF, airway epithelial cells are more susceptible to bacterial and viral
infection due to impairment of the host NO defense pathway. Polymorphisms of constitutive
NOS (nNOS and
eNOS) and reduced iNOS
expression contributes to decreased NO production along with
bacterial consumption , , , , . Alternatively, lower concentration of airway
NO in CF could be caused by lack of substrate,
L-arginine is a common substrate for both NOSs (e.g.,
iNOS) and arginase (ARG1).
ARG1 activity is increased in CF patients. Increased
ARG1 activity can result in
L-arginine deficiency and thereby contribute to low airway
NO formation and impaired pulmonary function , .
NO can also be released from
S-Nitrosoglutathione, an endogenous signaling molecule that
at low micromolar concentrations increases expression, maturation, and function of both
wild-type and deltaF508 CFTR in epithelial cells , , .
S-Nitrosoglutathione mainly acts independently of the
classic NO radical/cyclic GMP pathway . A
number of enzymes can catabolize S-Nitrosoglutathione to
form free NO radicals, including Cu/Zn superoxide dismutase
(SOD1) and thioredoxin reductases
(TXNRD1, TXNRD2 and
TXNRD3) , , , .
NO mediates many effects through production of cyclic GMP
(cGMP) by NO-sensitive guanylate cyclase.
cGMP activates type II isoform of cGMP dependent protein
kinase G (Protein kinase G 2). The latter phosphorylates
CFTR and increases its activity. NO-sensitive
Guanylate cyclase alpha-2/beta-1 isoform is localized in the
plasma membrane where it mediates stimulatory effect of NO
on CFTR activity much more efficiently than other
NO-dependent guanylate cyclase isoforms . The selective membrane effects
of the Guanylate cyclase alpha-2/beta-1 isoform in this
signaling relay are mediated by a compartmentalized pool of
cGMP that is resistant to degradation by cellular
phosphodiesterases, such as phosphodiesterase 5A (PDE5A)
, , .
NO and cGMP stimulate
CFTR activity and can down-regulate amiloride-sensitive
epithelial sodium absorption , , .
CFTR is an apical membrane Cl- channel in
epithelial cells. CFTR negatively regulates the
amiloride-sensitive epithelial sodium channel (ENaC).
ENaC inhibition by CFTR is
documented in CF patients which suffer from airway obstruction and chronic infection that
results from decreased mucociliary clearance secondary to missing
CFTR and high ENaC activity
CFTR and ENaC are the
principal rate-limiting steps for Cl- secretion and Na+ absorption
by ciliated airway epithelia. Mutations in the CFTR gene lead to hyperabsorption of
sodium chloride and a reduction in the periciliary salt and water content which leads to
impaired mucociliary clearance , , .
The mechanism of CFTR inhibition of
ENaC activity is not known. The proposed mechanisms range
from altered cellular trafficking of ENaC to direct
protein/protein interactions , , .
There is also a unique relationship in the CF airway between prokaryotic and
eukaryotic cells. The airway colonization with denitrifying bacteria, such as Pseudomonas
aeruginosa and Burkholderia species, alters nitrogen balance in the CF airway. CF lung
infection is associated with decreased concentrations of oxidized forms of
nitrogen (such as
NO) and increased
concentrations of reduced forms of nitrogen (such as nitrous
oxide (N(2)O) and ammonium
(NH(3))) in the CF airways .
The reduction of NO to N(2)O
can be catalyzed by enzymes like Nitric oxide reductase (NOR (P.
aeruginosa)) in the P. aeruginosa denitrification pathway , , . NOR (P. aeruginosa) can
also catalyze the reduction of N(2)O to molecular nitrogen
(N2) , . One of the most
prevalent species of the Burkholderia cepacia complex found in CF patients, Burkholderia
vietnamiensis, can produce a Nitrogenase
complex that catalyzes the reduction of
N(2) to NH(3) .
In epithelial cells, NH(3) inhibits chloride transport
(via CFTR channel), and NO
inhibits amiloride-sensitive sodium transport (via ENaC
channel) and augments chloride transport , , . Thereby, a shift from oxidized to reduced forms of nitrogen can increase
epithelial salt transport abnormalities in CF airways and adversely affect CF
Mucoid, mucA mutant P. aeruginosa bacteria cause chronic lung infections in CF
patients and are refractory to phagocytosis and antibiotics. As chronic CF lung disease
progresses, mucoid, alginate-overproducing strains emerge and become the predominant form
P. aeruginosa is capable of anaerobic growth by respiration using nitrite
(NO(2)(-)) as terminal electron acceptor. The reduction of
(NO(2)(-)) to NO is catalyzed
by P. aeruginosa enzyme Nitric reductase (NirS (P.
Mucoid, mucA mutant P. aeruginosa has the inherently low
NirS and NOR activity and,
thereby, has limited capacity for NO(2)(-) and
NO removal. Treatment of mucoid, mucA
mutant bacteria with NO(2)(-) (15 mM) at pH
6.5 under anaerobic conditions, similar to conditions within mucopurulent secretions in
the airways of CF patients, leads to the death of these bacteria. Thereby, the treatment
(e.g. aerosolization) of CF patients with NO(2)(-) and
NO can exert antimicrobial effect on mucoid P. aeruginosa
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